The cornea is the primary refractive element of the eye and therefore it must be smooth and transparent for good vision. Transparency is most affected by the hydration level of the stromal connective tissue. Hydration is dependent on the metabolic activities of the corneal epithelium and endothelium;however it is the endothelium that is primarily responsible for pumping ions and fluid that maintains the steady-state hydration of the stroma. The long-term goal of this project is to understand how the corneal endothelium does this job, so that medical therapies can be developed to enhance endothelial function in corneas that have been compromised by disease or trauma. The endothelial ion and fluid pump is most dependent on HCOa" for its function. Our approach has been to identify, characterize, and integrate the membrane pumps, transporters and channels into a model for transendothelial HCCV and fluid transport in a cultured endothelial cell system. In this period of the project we focus on understanding the mechanisms for apical HCOa'efflux, particularly the roles of carbonic anhydrases (CAs) and anion exchangers and how they may buffer and facilitate the transport of lactic acid. To do this, we will identify and localize lactate: H+ cotransporters and determine if lactate fluxes are reduced by pharmacological inhibitors for CAs and HCO3" transporters as well as following treatment with small interfering RNA to reduce CA or transporter expression. We also examine the roles of adenosine receptor stimulation and soluble adenylyl cyclase in setting cell [cAMP], which has a stimulatory effect on endothelial function. This will also use the siRNA approach and examine cAMP/PKA mediated phosphorylation of transporters, myosin light chain, and CREB transcription factor as well as the effects on net HCO3" transport as measures of relative PKA activity. Lastly, we will examine the model of HCOa" transport that is being built in vitro with cultured cells in the in vivo rabbit cornea. Our approach is to use lentiviral delivery of interfering RNA molecules to reduce specific transporter expression in vivo and examine the effect on corneal thickness and endothelial function. The results from this Aim will tell us the relative importance of the various transporters in maintaining corneal hydration in the in vivo cornea.